This chapter reviews applications of electrospray ionization (ESI) to the analysis of RNA and its constituents. Some examples of modified nucleosides discovered by liquid chromatography (LC)/mass chromatography (MS) and characterized include four sugar-methylated nucleosides in tRNA from hyperthermophilic archaea, 5-methylcarboxymethyl-2’-O-methyluridine in rat liver tRNASer[Sec] *, and two sugar-methylated nucleosides in the cap4 subunit of trypanosomal mRNA. A method has been implemented for mapping modified nucleosides in RNA, based on electrospray ionization mass spectrometry of RNase T1-derived oligonucleotides. The ability to mass measure the small peaks suggests that oligonucleotides can be analyzed at levels lower than the 20 pmol of tRNA utilized in the present example. To examine levels of modified nucleosides in tRNA as a function of growth temperature in the thermophilic archaeon Pyrococcus furiosus, it was grown at 70, 85, and 100°C. The strategies and examples of nucleic acid analysis using mass spectrometry presented in this chapter utilize ESI because of its successful implementation for the analysis of nucleic acids and their constituents from the monomer level in digests all the way up to intact tRNA and 5S rRNA.

Expanded UV chromatogram (A) and reconstructed ion chromatograms for the MH+ ion of methylguanosine (B) (m/z 298) and the BH+2 ions for methylguanine (C) (m/z 166) and guanine (D) (m/z 152), extracted from the analysis shown in Fig. 2. For clarity, ion abundances have been normalized to the most abundant peak in each panel.

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10.1128/9781555818296/fig3-3.gif

Figure 3

Expanded UV chromatogram (A) and reconstructed ion chromatograms for the MH+ ion of methylguanosine (B) (m/z 298) and the BH+2 ions for methylguanine (C) (m/z 166) and guanine (D) (m/z 152), extracted from the analysis shown in Fig. 2. For clarity, ion abundances have been normalized to the most abundant peak in each panel.